J. Phycol. *, ***–*** (2018) © 2018 Phycological Society of America DOI: 10.1111/jpy.12766

BAFFINELLA FRIGIDUS GEN. ET SP. NOV. (BAFFINELLACEAE FAM. NOV., ) FROM BAFFIN BAY: MORPHOLOGY, PIGMENT PROFILE, PHYLOGENY, AND GROWTH RATE RESPONSE TO THREE ABIOTIC FACTORS1

Niels Daugbjerg,2 Andreas Norlin Marine Biological Section, Department of Biology, University of Copenhagen, Universitetsparken 4, Copenhagen Ø DK-2100, Denmark and Connie Lovejoy Departement de Biologie, Universite Laval, 1045 avenue de la Medecine, Quebec, Quebec, G1V 0A6, Canada

Twenty years ago an Arctic cryptophyte was Abbreviations: BA, Bayesian analysis; BS, bootstrap isolated from Baffin Bay and given strain number support; Cr-PC, cryptophyte-phycocyanin; Cr-PE, CCMP2045. Here, it was described using cryptophyte-; ML, maximum likeli- morphology, water- and non-water soluble pigments hood; PP, posterior probability and nuclear-encoded SSU rDNA. The influence of temperature, salinity, and light intensity on growth rates was also examined. Microscopy revealed = typical cryptophyte features but the Cryptophytes ( ) are ubiquitous in color was either green or red depending on the marine and freshwater ecosystems worldwide and a few have been recorded to form blooms light intensity provided. Phycoerythrin (Cr-PE 566) was only produced when cells were grown under (e.g., Laza-Martınez 2012, Supraha et al. 2014, and low-light conditions (5 lmol photons m 2 s 1). references therein). However, they also reside in Non-water-soluble pigments included chlorophyll a, more extreme environments, for example, soil (Paulsen et al. 1992), snow (Javornicky and Hindak c2 and five major carotenoids. Cells measured 8.2 3 5.1 lm and a tail-like appendage gave them a 1970), and inside ikaite columns (Ikka fjord, South- comma-shape. The nucleus was located posteriorly west Greenland; Kristiansen and Kristiansen 1999, and a horseshoe-shaped chloroplast contained a N. Daugbjerg, unpub. data). The currently accepted single . Ejectosomes of two sizes and a of Cryptomonada includes two classes: anterior to the pyrenoid were the photoautotrophic Cryptophyceae (note: Chilomo- discerned in TEM. SEM revealed a slightly elevated nas is heterotrophic but this state is most likely sec- vestibular plate in the vestibulum. The inner ondarily derived) and the phagotrophic periplast component consisted of slightly Goniomonadea. Until recently, the latter class con- overlapping hexagonal plates arranged in 16–20 sisted of only a single and some oblique rows. Antapical plates were smaller and environmental sequences, but Shiratori and Ishida their shape less profound. Temperature and salinity (2016) have described the second heterotrophic studies revealed CCMP2045 as stenothermal and (Hemiarma marina). euryhaline and growth was saturated between 5 and The species diversity of Cryptophyceae has been 20 lmol photons m 2 s 1. The phylogeny based studied for more than 180 years as Ehrenberg dis- on SSU rDNA showed that CCMP2045 formed a covered the first cryptophyte ( ovata)in distinct clade with CCMP2293 and Falcomonas sp. 1831. However, the first detailed descriptions and isolated from Spain. Combining pheno- and drawings were published 7 years later (Ehrenberg genotypic data, the Arctic cryptophyte could not be 1838). According to AlgaeBase, as of May 2018, placed in an existing family and genus and there are 41 genera listed in the Cryptophyceae (Guiry and Guiry 2018) and of these 21 are mono- therefore Baffinellaceae fam. nov. and Baffinella = frigidus gen. et sp. nov. were proposed. typic ( 53.7%). At the biochemical level, cryptophytes possess a Key index words: Arctic flagellates; cryptophytes; unique combination of pigments that include growth rates; phylogeny; taxonomy; ultrastructure chlorophylls a and c2, one of two and the cryptophyte specific carotenoid alloxanthin, alongside other common carotenoids (Hill and Rowan 1989, Cerino and Zingone 2007, Egeland 1 Received 17 April 2018. Accepted 27 June 2018. First Published 2016). The two phycobiliproteins found in crypto- Online 25 July 2018. 2Author for correspondence: e-mail [email protected]. phytes are phycoerythrin and phycocyanin, and Editorial Responsibility: L. Graham (Associate Editor) these have different structural configurations

1 2 NIELS DAUGBJERG ET AL. identified by their absorption peaks. Some of these with existing families. Thus, a new family Baffinel- configurations are unique (Hill and Rowan 1989, laceae fam. nov. was also described. Novarino 2012). For each genus, only one type of is present except for that has been shown to contain species with either MATERIALS AND METHODS Cr-PC 615 and Cr-PE 555 (Hill and Rowan 1989). Culture. Strain CCMP2045 was isolated from a water sam- 0 As some families possess the same phycobiliprotein ple collected in Baffin Bay (June 23, 1998; 76°19 15″ N, ° 0 ″ (Clay et al. 1999), it has been discussed if the pho- 75 49 02 W; Fig. 1) at a depth of 11 m. A sub-culture of this tosynthetic pigment profile can be used to distin- strain was sent to the Marine Biological Section at University of Copenhagen where it has been kept at 4°C in L1 medium guish taxa at the family level (Fritsch 1935, (Guillard and Hargraves 1993) with a salinity of 30 and a Pringsheim 1944, Butcher 1967, Hill and Rowan light:dark cycle of 16:8 h. 1989). Microscopy. Live cells were studied using a Carl Zeiss Axio At the light microscopical level, the gross mor- Imager.M2 with a 639 oil immersion lens. Micrographs were phology of cryptophytes is somewhat similar: droplet taken with a Zeiss AxioCam HRc digital camera (Zeiss, Ober- shape and two flagella that emerge from the kochen, Germany). Chlorophyll fluorescence was recorded subapical end. Thus, most taxonomic differences using a Zeiss AxioCam MRc digital camera and the filter set 09 (excitation BP450-490, emission LP515). Length and width relate to the ultrastructure of the periplast compo- of 59 cells were measured using Zen image acquisition soft- nent, position of the nucleomorph, appearance of ware from Zeiss. Means and standard deviations were calcu- the rhizostyle, and the furrow-gullet complex (Clay lated using IBM SPSS statistics (ver. 24, Armonk, NY, USA). et al. 1999). For transmission electron microscopy, 5 mL of a dense cul- In the Arctic, cryptomonads have been shown to ture was fixed in 5 mL of 4% glutaraldehyde in 0.2 M cacody- contribute to both pelagic and sympagic communi- late buffer containing 0.5 M sucrose for 1 h and 25 min. The ties of microalgae. They are also part of the spring fixed cells were pelleted by centrifugation and the super- natant except for 2 mL was removed. Two milliliter 0.2 M bloom when the sea ice melts and nutrients are cacodylate buffer containing 0.5 M sucrose was added. After released to the open water column (Mikkelsen 20 min, 2 mL of the supernatant was removed and replaced et al. 2008, Vallieres et al. 2008, Poulin et al. 2011, with 2 mL 0.2 M cacodylate buffer without sucrose for Coupel et al. 2012). However, very few studies have 20 min. The mix was centrifuged at 1,201 g for 10 min after addressed the taxonomy of Arctic cryptophytes. each of these steps. For post-fixation 2 mL of the supernatant Recently, the species diversity of Arctic marine pro- tists including cryptomonads was studied by molec- ular techniques (e.g., clone libraries and high throughput sequencing; Sørensen et al. 2012, Mar- quardt et al. 2016, Stecher et al. 2016). These stud- ies have provided detailed information on operational taxonomic units, but for many of the determined ribotype sequences we have little understanding of the corresponding morphos- pecies. In this study, a cryptophyte was isolated from material collected in Baffin Bay in Arctic Canada (1998), and later deposited at Provasoli-Guillard National Center for Marine Micro Algae and Micro- biota in Maine, USA (Potvin and Lovejoy 2009), where it was given the strain number CCMP2045. The aim of this study was to describe this strain using a polyphasic approach, which included a mor- phological characterization using LM, TEM and SEM. The composition of both water and non-water- soluble pigments was analyzed to describe the pro- file of photosynthetic marker pigments. To infer the phylogeny of CCMP2045, the nuclear-encoded SSU rDNA sequence was compared to 50 other cryp- tomonads. Finally, growth rates were estimated through autecological experiments elucidating salin- ity and temperature tolerance limits as well as growth rates at different light intensities. Based on these results, a new genus and species Baffinella frigi- dus gen. et sp. nov. was proposed to accommodate for strain CCMP2045. The combination of morpho- FIG. 1. Map of sampling location (*)ofBaffinella frigidus gen. logical features and phycobiliproteins did not fit et sp. nov. in Baffin Bay. BAFFINELLA FRIGIDUS, AN ARTIC MARINE CRYPTOPHYTE 3

was removed and 2 mL, consisting of 0.5 mL 4% OsO4 and previously been determined by Potvin and Lovejoy. The 1.5 mL 0.2 M cacodylate buffer was added for 1 h before sequences were retrieved from GenBank (accession number being rinsed with 0.2 M cacodylate buffer. For dehydration, a GQ375264 and GQ375265, respectively) and added to an series of ethanol was used, 20 min for each concentration alignment consisting of 50 other SSU rDNA sequences of (30%, 50%, 70%, 90%, and 100% in molecular sieves) with cryptomonads including 18 genera and 38 taxa identified to 100% in molecular sieves used twice. The pellet was rinsed species. The data matrix was edited using the JALVIEW twice in propylene oxide before embedding and left over- sequence editor (ver. 2.9.0b2; Waterhouse et al. 2009) follow- night in a mix of 1:1 propylene oxide and Spurr’s resin. The ing an alignment based on ClustalW as implemented in the next day Spurr’s resin was replaced by fresh Spurr’s resin for software. The data matrix consisted of 1942 base pairs includ- 6 h before the pellet was embedded in a cast with fresh ing introduced gaps. Phylogenetic reconstructions were based Spurr’s resin and polymerized at 70°C overnight. Thin sec- on BA and ML analyses. BA was performed using MrBayes tions were cut with a diamond knife and stained for transmis- (ver. 3.2.5 X64; Ronquist and Huelsenbeck 2003) on a desk- sion electron microscopy using uranyl acetate and lead top computer where 5 9 106 generations were run and a tree citrate. The microscope used was a JEM 1010 electron micro- was sampled every 1,000 generations. The burn-in value was scope (JEOL Ltd., Tokyo, Japan) and micrographs were taken evaluated using the lnL value plotted as a function of genera- using a GATAN Orius SC1000 digital camera (Gatan, Pleasan- tions. The lnL values converged after 101,000 generations ton, CA, USA). The accelerating voltage was 80 kV. and 101 trees were discarded, leaving 4,900 trees for generat- For scanning electron microscopy, 3.5 mL of the culture ing a 50% majority-rule consensus tree in PAUP* (Swofford was fixed in 3.5 mL 4% glutaraldehyde in 0.2 M cacodylate 2002). ML analysis was run using the PhyML software (Guin- buffer containing 0.5 M sucrose for 1.5 h, adding 1 mL of don et al. 2010) on a desktop computer with the parameter 4% OsO4 in seawater after 10 min. The cells were placed on settings obtained from jModelTest (ver. 2.1.3; Darriba et al. a5lm isopore filter and rinsed in 0.2 M cacodylate buffer 2012). GTR+I+G was chosen as the best fit model for the data containing 0.5 M sucrose followed by 0.2 M cacodylate buffer matrix (gamma = 0.568 and p-invar = 0.464). BS were calcu- containing 0.25 M sucrose and finally 0.2 M cacodylate buffer lated from 500 replications and mapped on the tree from BA without sucrose, for 10 min each. Dehydration was done with alongside the PP values. an ethanol series (30%, 50%, 70%, 90%, and 99.9%) for The was used to root the 10 min each. Dehydration was finished with 100% ethanol in tree as Burki et al. (2012) in a large-scale phylogenetic study molecular sieves for 30 min twice before the sample was criti- had shown this taxon to form a sister to the cryptomonads. cal-point-dried. The isopore filter was attached to an alu- Autecological experiments. For salinity and temperature, the minum stub and sputter coated with gold/palladium in a autecological experiments were performed in 60 mL Nunc JEOL JFC-2300HR sputter coater before being observed in a flasks. Triplicate flasks were placed in 24 L glass tanks con- JEOL JSM-6335F field emission scanning electron microscope taining demineralized water, circulated by an aquarium with a secondary electron detector and running at 12 kV pump. A cooling system controlled by a custom made relay (JEOL Ltd.). switched cooled water on/off and thus ensured accurate con- Pigment analyses. Extraction of the water-soluble pigment trol of pre-set experimental temperatures (0.1°C). For light (phycobiliprotein) was based on Lawrenz et al. (2011) and experiments, the Nunc flasks were placed in a 4°C walk-in cli- briefly described here. An exponentially growing live sample mate room, at areas where the desired light intensities were (5–10 mL) was centrifuged for 5 min at 3075 g and the marked. Light measurements were made using a spherical supernatant was removed. A volume of 2.5 mL of PBS buffer PAR sensor (Walz, ULM-500; Heinz Walz GmbH, Effeltrich, (pH 7.2 or 7.5) was added and cells were disrupted by two Germany). The light experiments were conducted at a light:- freeze-thaw cycles (i.e., 10 min at 80°C followed by thawing dark cycle of 16:8 h, because a shared climate room was used. at room temperature). The extraction lasted at least 20 h at pH was measured regularly (often every second day) to moni- 4°C. Finally, a subsample of 1.2 mL was put in a 1.5 mL tor increases due to photosynthetic activity. All experiments Eppendorf tube and centrifuged for 15 min at 12,202g. The used a start concentration of ~1,000 cells mL 1. Samples for supernatant was analyzed by scanning the spectrum between cell abundance measurements were collected shortly after the 350 and 800 nm in a Shimadzu UV180 spectrophotometer start of the experiments (time = t ). Due to a linear correla- 0 (Holm & Halby A/S, Brøndby, Denmark). Extraction of non- tion between fluorescence of chlorophyll a and cells mL 1 water-soluble pigments for HPLC were extracted in 95% when grown at the same light intensity, we converted fluo- methanol, and separated with a Accela 600 HPLC system rometer readings to cell abundances based on a standard (Thermo Scientific, San Jose, CA, USA) on a reverse-phase curve. See Figure S1 in the Supporting Information for the Hypersil Gold C-8 column and a solvent gradient containing standard curve used in the temperature and salinity experi- methanol, aqueous pyridine, acetone, and acetonitrile (Zap- ments and Figure S2 in the Supporting Information for the ata et al. 2000). An Accela PDA detector recorded chro- standard curves used in the light experiments. Samples were matograms at 450 nm and pigment spectra over the taken every second day by pipetting 1,140 lL from each repli- wavelengths 350–800 nm. A Finnigan Surveyor FL Plus fluo- cate into 1.5 mL Eppendorf tubes. Raw fluorescence units rescence detector with excitation of 440 nm and emission at were measured using a Trilogy fluorometer equipped with 650 nm (optimized for Chl a) was also used to identify and the blue module (Trilogy, Turner Designs Instruments, Sun- quantitate Chl pigments. The system was calibrated by nyvale, CA, USA). The first replicate (A series) of each treat- repeated injections of 30 pigment standards (Sigma Aldrich ment was used to calculate standard curves by fixing it with (St. Louis, MO, USA) or DHI (Hørsholm, Denmark) LAB Lugol’s iodine (5% final concentration) and counting cell products). ChromeQuest software was used to identify and numbers using 1.0 mL Sedgewick Rafter chambers (Pyser-SGI quantitate the concentration of the pigments. The photodi- Limited, Edenbridge, UK). ode array spectrum of each peak was checked against the ref- Temperature experiments. The growth rates were examined erence spectra of the standard or against the reference at 4.0°C, 6.0°C, 8.0°C and 10.0°C and at a salinity of 35. spectra in Roy et al. (2011) for pigments for which there are The light intensity was 100 lmol photons m 2 s 1 and no standards. the light:dark cycle 24:0 h to simulate the Arctic summer Phylogenetic inference. The phylogenetic inference of conditions. CCMP2045 was based on the nuclear-encoded SSU rDNA Salinity experiments. These were conducted at salinities of gene, alongside the sequence of CCMP2293, which had 0, 5, 10, 15, 20, 25, and 30 and all at 4.0 0.1°C. An 4 NIELS DAUGBJERG ET AL. acclimation period of 3 d was included at salinities of 10 used side; flagella inserted at one-third cell length for experiments at 0, 5, and 10, respectively; 20 used for from the apex. experiments at 15 and 20, respectively; and 30 used for exper- Etymology: Frigidus from latin meaning cold, refer- iments at 25 and 30, respectively. The light setting was identi- cal to that used in the temperature experiments. The same ring to its Arctic origin. set of flasks and therefore the same data were used for the Holotype: An epon-embedded sample of strain temperature experiment at 4°C and salinity experiment at 35. CCMP2045 has been deposited at the National His- Light experiments. These were conducted at 5, 20, 40, 60, tory Museum, University of Copenhagen (C), acces- 80, and 100 lmol photons m 2 s 1 at 4°C and a salinity of sion no. C A92087. 30. The light:dark cycle was 16:8 h. GenBank accession number: GQ375264, represents Calibration of growth rates. Growth rates were calculated for the nuclear-encoded SSU rDNA sequence. each replicate and the mean was based on three replicates Authentic culture: CCMP2045 at the Provasoli-Guil- (n = 3). The maximum growth rate (l) was defined as the number of cell divisions d 1 and calculated as: lard National Center for Marine Algae and Micro- biota.  Type locality: Baffin Bay (76°19015″ N, 75°49002″ ln Nt2 l ¼ Nt1 W), Arctic Canada. t2 t1 Morphology, color, and swimming behavior in LM. Cells measured 5.6–11 lm in length (average: where Nt1 and Nt2 were the cell abundances at the start (t1) l = – l and end (t ), respectively. 8.2 1.2 m, n 59) and 4.3 6.1 m in width (av- 2 l = – Statistics. Comparisons of mean growth rates by one-way erage: 5.1 0.4 m, n 59; Fig. 2, A J). Most cells ANOVA tests were performed using Graphpad Prism (ver. 7) had a comma-shaped appearance due to a small and GraphPad Prism (ver. 6, GraphPad Software, La Jolla, tail-like appendage in the antapical end, others were CA, USA) was used to plot the data and do regression analy- bluntly rounded (Fig. 2, A, B and D). In optical ses. cross-section, cells were spherical (Fig. 2J). Two unequally long flagella were inserted in an apical RESULTS depression (the gullet, Fig. 2, A-C, F, H and K). Rows of large ejectosomes were observed close to We propose the following taxonomy for strain the gullet (Fig. 2, A, B and E). A large was CCMP2045. present in the apical end (Fig. 2H), whereas the Class Cryptophyceae. Order . nucleus was located in the antapical end (Fig. 2, C with phycobiliprotein Cr-phycoerythrin and G). The structure of the periplast component 566 (Cr-PE III), present in some. surrounding the cell could be discerned in the light Description: Baffinellaceae Daugbjerg & Norlin microscope (Fig. 2F). The single chloroplast was fam. nov. seen to extend in almost the full length of the cell Furrow-gullet complex present, without stoma; (Fig. 2M). In cross-section, the two chloroplast lobes nucleomorph positioned anterior to the pyrenoid; formed a horseshoe-shape (Fig. 2, J and L). A single inner periplast component with partly overlapping pyrenoid was observed in connection with the hexagonal plates; subapical vestibular plate present; chloroplast and it was surrounded by a starch sheet rhizostyle present. (Fig. 2I). The pyrenoid was located in the middle of Description: Baffinella Norlin & Daugbjerg gen. the cell (Fig. 2G). A refractive crystal-like structure nov. was present in many cells (Fig. 2, A–C). The ejecto- Two unequal flagella protruding from a furrow-gul- somes discharged when cells experienced elevated let system in the anterior end; vestibular plate located heating from prolonged observations in the light on dorsal side of gullet; a single horseshoe-shaped microscope (Fig. 2N). Live cells swam while rotating chloroplast with a single pyrenoid surrounded by a around their longitudinal axis. Depending on the starch sheet; apical end rounded and antapical end light intensity provided the color of CCMP2045 was acute or bluntly rounded; hexagonal periplast plates either green or red. Live cells grown at low light less profound in shape at the antapical end. (5 lmol photons m 2 s 1) were red (Fig. S3, A– Type species: B. frigidus sp. nov. C in the Supporting Information), whereas cells Etymology: from the name of the collection area, were green if grown at light intensities ≥10 lmol the Baffin Bay, with the Latin, feminine suffix ella, photons m 2 s 1 (Fig. S3, D–F). Interestingly, meaning small. CCMP2045 has been identified as sp. in Description: Baffinella frigidus Norlin and Daugb- the culture collection at Bigelow Laboratory for jerg sp. nov. Ocean Sciences (http://ncma.bigelow.org/cc Comma-shaped in lateral view, spherical in mp2045); probably due to low light conditions pro- cross-section; dorsal side convex, ventral side vided. more straight; live cells 5.6–11 lmlongand4.3– l External morphology in SEM. The apical vestibulum 6.1 m wide; gullet deep, lined with rows of large was part of a furrow-gullet complex (Fig. 3, A, B ejectosomes; pyrenoid bisected by cytoplasmic and D). The two flagella emerged on the right side tongue; mid-dorsal band present at antapical end; of the gullet (Fig. 3, A and B). The vestibular plate nucleus in antapical end, slightly to the dorsal formed a tongue-like structure located in the upper BAFFINELLA FRIGIDUS, AN ARTIC MARINE CRYPTOPHYTE 5

2 1 FIG. 2. Light microscopy of Baffinella frigidus gen. et sp. nov. grown at 100 lmol photons m s .(A–J, N) Nomarski interference contrast; (K) phase contrast; (L, M) epiflourescence. (A–C) Ventral view of cell showing gullet with large ejectosomes (e) and chloroplast (c). Note tail-like appendage (arrow, B) and nucleus (N, C) in the antapical end. Refractive vesicles are visible in A-C (arrow heads). (D– E) Lateral view showing marked comma shape in some cells (D) and bluntly rounded antapical ends in other cells (E). (F) Lateral view revealing the arrangement of periplast plates. (G) Latero-ventral view displaying posterior nucleus (N), and central pyrenoid (p). (H) Lat- eral view showing cell with large vacuole (v) in apical end. (I) Pyrenoid (p) surrounded by a large starch sheet (s). (J) Optical cross-sec- tion of partial chloroplast forming a horseshoe-shape. (K) Lateral-ventral view showing unequally long flagella (df, dorsal flagellum; vf, ventral flagellum). (L, M) Chloroplast in cross-section (L) and lateral view (M). Note horseshoe-shaped chloroplast in L. (N) Disintegrated cell with discharged ejectosomes (e) twice the cell in length. part of the gullet toward the dorsal side (Fig. 3, A, prominent in the antapical end where they clus- B, D, F, and G). The periplast component consisted tered more closely compared to the rest of the cell. of slightly overlapping hexagonal plates arranged in Discharged ejectosomes were seen to penetrate the 16–20 oblique rows (but see below). Each plate was cell perimeter (Fig. 5, D and E). The periplast was ~0.6 lm in length. The periplast plates in the tail- serrated giving cells a coarse appearance in sec- like antapical end were smaller, the shape less pro- tioned material (Figs. 4A and 5A). A single chloro- found and the number of rows (if any) could not plast containing a pyrenoid was present on the be determined (Fig. 3, A–C). The tail-like appen- dorsal side of the cell (Figs. 4A and 5A). The pyre- dage had bent in Figure 3, A, B, and D, whereas it noid was surrounded by a starch sheet (Figs. 4, A, was pointed in Figure 3C. A mid-dorsal band (sensu C, F and 5A) and the pyrenoid matrix itself was Hill 1991a) in the antapical end could also be dis- bisected by a cytoplasmic tongue (Figs. 4F and 5A). cerned (Fig. 3H). A drawing illustrating the general Flagellar basal bodies and a few features of the flag- cell outline, extent of the furrow, the vestibular ellar apparatus could be observed (Fig. 4, C–E). A plate in the vestibulum and the appearance of 3-dimensional reconstruction was not attempted but hexagonal periplast plates (lower part only) and the striated rootlet (Fig. 4, D and E), the striated how these become smaller toward the antapical end rootlet (Fig. 4E) and a short, non- was shown in Figure 6A. keeled rhizostyle (Fig. 4C) could be observed. The Ultrastructure in TEM. The general ultrastructure vestibular plate was found to be in association with and disposition of cell was illustrated in the dorsal part of the gullet (Fig. 4, B and D), but a Figure 4, A and B. Large, regularly spaced ejecto- detailed understanding of the ultrastructure needs some vesicles were prominent on the ventral side of further studies. The nucleus was located in the the cell (Fig. 4A) and lined the inside of the gullet antapical end of the cell (Fig. 4, A and B), whereas (Figs. 4B and 5, A and B). Small ejectosome vesicles the nucleomorph was found anterior to the pyre- were seen just underneath the periplast plates noid toward the apical end (Fig. 4F). The Golgi (Figs. 4A and 5, A–C). They were especially body was positioned just underneath the vestibulum 6 NIELS DAUGBJERG ET AL.

FIG. 3. Scanning electron microscopy of Baffinella frigidus gen. et sp. nov. illustrating external features. (A) Ventral view of cell with two flag- ella protruding from the right side of the furrow-gullet complex. Note discharged ejectosomes. In the gullet, the vestibular plate (vp) is seen as a tongue-like structure. (B) Ventro-lateral view with vestibular plate (vp) in the anterior part of the vestibulum. Note bending of antapical end in A and B. (C) Lateral view illustrating overlapping hexagonal periplast plates. Note that periplast plates get smaller toward antapical end. (D) Ventral view showing the furrow (fu) extending from the vestibulum. (E) High magnification the slightly overlapping periplast plates. (F) High magnification of vestibular plate (vp); same cell as in B. (G) Dorsal view of the vestibular plate (vp) raising above the apical end. (H) High mag- nification of tail-like appendage showing mid-dorsal band (arrow heads) and smaller periplast plates toward antapical end. BAFFINELLA FRIGIDUS, AN ARTIC MARINE CRYPTOPHYTE 7

FIG. 4. Transmission electron microscopy of Baffinella frigidus gen. et sp. nov. (A) Lateral view showing large ejectosomes (le), posterior nucleus (N), chloroplast (c) and pyrenoid (p) with encircling starch sheet and anterio-ventrally positioned Golgi body (G). Note small ejectosomes (se) near adjoining, slightly raised anterior part of the periplast plates. (B) Lateral view showing furrow (fu) and gullet (gu) lined by large ejectosomes (le). Vestibular plate (vp) is in association with subapical part of the cell. Position of nucleus (N), pyrenoid (p), starch sheet (s) and chloroplast (c) also indicated. (C) Oblique section through apical end showing flagellar basal bodies, a short non-keeled rhizostyle (r) and pyrenoid (p) surrounded by starch (s). Large ejectosome (le). (D) Longitudinal section through apical end showing ventral (VB) and dorsal (DB) basal bodies with striated rootlet (SR). (E) Higher magnification of D showing striated rootlet (SR), striated fibrous root associated microtubular root (SRm) and distal (DS) and proximal septum (PS) of the flagellar transition zone. Ventral (VB) and dorsal (DB) basal bodies also indicated. (F) Longitudinal section showing nucleus (N) toward the antapical end, bisected pyrenoid (p) surrounded by a starch sheet (s) and anteriorly positioned nucleomorph (nm). Note also parietal chloroplast (c). 8 NIELS DAUGBJERG ET AL. and adjacent to the basal bodies (Fig. 4F). A draw- Autecology. The growth potential of ing illustrating the internal position of cell orga- Baffinella frigidus to changes in temperature, salin- nelles was shown in Figure 6B. The single ity, and light intensity was evaluated by one horseshoe-shaped chloroplast reaches from the api- parameter experiments (Fig. 9, A–C). Following cal end and down to the anterior end of the our experimental setup, the Artic cryptophyte nucleus in the antapical end of the cell. CCMP2045 was able to grow (divide) at tempera- Pigment profile. The absorption spectrum of water- tures ranging from 4°Cto8°C. No growth was soluble pigments revealed a peak between 562 and observed at 10°C (Fig. 9A). Significant differences = < 566 nm (Fig. 7). This indicated the presence of phy- in growth rates (F2,6 415.5; P 0.0001; one-way cobiliprotein Cr-PE 566. Analysis of non-water sol- ANOVA) were found between treatments at 4°C, uble pigments revealed the presence of chlorophylls 6°C, and 8°C, respectively, as indicated by lower a and c in addition to common carotenoids like case letters (a-c). The highest growth rate was 2 alloxanthin, crocoxanthin, b,e-carotene, lutein, and obtained at 4°C with 0.40 divisions d 1 and the zeaxanthin (data not shown). Among the non-water- lowest growth was obtained at 8°C with 0.15 divi- soluble pigments chlorophyll a and alloxanthin sions d 1. This equals a 2.7 times reduction in were present with almost equal amounts (data not growth rate. shown). The results of the salinity one parameter experi- Phylogeny and sequence divergence. The deepest ment revealed that Baffinella could grow at salinities branches of the phylogeny based on SSU rDNA ranging from 5 to 35 (Fig. 9B). The highest growth were generally not supported from PP or BS values was obtained at a salinity of 20 (0.45 divi- (Fig. 8). Hence, the relationship and evolutionary sions d 1), and the lowest was obtained at a salin- history among lineages, which often corresponded ity of 5 (0.30 divisions d 1). The one-way ANOVA = to families of cryptophytes, could not be discerned. (F6,14 14.94) showed three statistically different However, the lineages comprising families were for groups labelled a-c in Figure 9B and that the growth the most part highly supported and in accordance rate at a salinity of 20 was significantly higher with the currently accepted classification (Clay et al. (P < 0.05) compared to that at salinities of 5, 10, 1999, Clay 2015) but with the addition of Falcomon- and 30, but not significantly higher (P > 0.51) com- adaceae (see Discussion). This was evident from pared to salinities at 15, 25, and 35. The growth rate superimposing cryptophyte families onto the phy- at a salinity of 5 was significantly lower (P < 0.01) logeny that included 18 genera. Interestingly strains compared to all other salinities (Fig. 9B). CCMP2045 (studied phenotypically here) and The light experiments demonstrated that the CCMP2293 also isolated from Baffin Bay, but col- growth rates were not significantly different when lected 10 April 1998, had identical SSU rDNA grown at 20 lmol photons m 2 s 1 and above sequences. These strains formed an unresolved but (Fig. 9C). Here, the growth rate was 0.39 divi- highly supported clade (PP = 1.0 and BS = 100%) sions d 1, whereas the growth rate at 5 lmol pho- with a strain identified as Falcomonas sp. from North tons m 2 s 1 was significantly (P < 0.001) lower Western Spain (Raho et al. 2014). CCMP2045/ and only reached 0.17 divisions d 1. The one-way CCMP2293 and the Spanish isolate of Falcomonas sp. ANOVA (F = 130.7) showed two groups, thus, 5,12 only differed by 1 transition (C↔T) in the 1,686 one with 5 lmol photons m 2 s 1 and the other base pairs included in the comparison. Thus, a very containing the rest of the light intensities (labelled low sequence divergence (0.06%) was estimated a and b, respectively, in Fig. 9C). Hence, between these taxa. Despite the clades comprising Baffinella frigidus was light saturated a 20 lmol pho- I and II (see Fig. 8) formed sister tons m 2 s 1 and may be even at slightly lower groups to Baffinella (CCMP2045, CCMP2293) and levels but this has to be studied in more detail. “Falcomonas sp.” this relationship was not statistically For all autecological experiments, the growth ter- supported (PP = 0.88, BS <50%). The heterotrophic minated when the pH reached 8.6–9 (data not genus Goniomonas (with three species included shown). Graphs for all autecological experiments here) within the family Goniomonadaceae branched showing cell abundances as a function of time are of as the first cryptomonad lineages (not mono- provided as supplementary Figures S4–S6 in the phyletic in Fig. 8). Geminigeraceae formed four Supporting Information. highly supported but polyphyletic clades if we include Urgorri within this family (labeled I to IV in Fig. 8). The early divergence of the phycoerythrin DISCUSSION containing marine Urgorri complanatus was not statis- To the best of our knowledge, this is the first mar- tically supported even though it did branch of as ine cryptophyte described from high Arctic waters. the first autotrophic cryptophyte. The phycocyanin- The description was based on a polyphasic approach containing cryptophytes seemed to form a mono- that included pheno- and genotypic characters in phyletic group but the support for this lineage was addition to exploring the potential growth rates at low from BA and lacking in ML bootstrap different temperatures, salinities and light intensi- (PP = 0.88, BS <50%). ties. BAFFINELLA FRIGIDUS, AN ARTIC MARINE CRYPTOPHYTE 9

FIG. 5. Transmission electron microscopy of Baffinella frigidus gen. et sp. nov. (A) Cross-section at the level of the bisected pyrenoid (p) enclosed by starch (s). The furrow (fu) and gullet (gu) complex also visible. Rows of large ejectosomes (le) located near the gullet. Note also single, parietal chloroplast (c). (B) Posterior part of cell showing gullet (gu), nucleus (N), large (le), and small (se) ejectosomes and horseshoe-shaped chloroplast (c). (C) Serrated outline of cell exterior is due to overlapping periplast plates. Chloroplast (c) sepa- rated from by small somewhat evenly distributed small ejectosomes (se). (D, E) Serrated outline of cell exterior and cell body traversed by discharged ejectosomes (de). Numerous small sized, non-discharged ejectosomes (se) also present.

Taxonomy and phylogeny. The taxonomy of the Chroomonadaceae (with two distinct lineages) and Cryptophyceae has been based on cell size and Geminigeraceae (with distinct four lineages if shape, furrow-gullet complex, inner, and outer com- accepting Urgorri as a member) were not. Hence, position of the periplast components, position of additional studies are needed to fully understand the nucleomorph and the type of biliprotein (either the taxonomy of the latter two families of Crypto- red or blue-green water-soluble pigments; for exam- phyceae. ple, Clay et al. 1999, Novarino 2012). Baffinella frigi- Vestibular plate. Baffinella had no single synapo- dus possessed morphological and biochemical morphic trait that separated it from other crypto- features typical of cryptophytes but also some phytes. Rather it had a unique combination of uncommon characters. Table 1 provides a list of characters that made it distinct. As can be seen from characters and character states currently used to dis- Table 1 this applies to the majority of cryptophytes. tinguish both marine and freshwater cryptophyte One of the more unusual features of Baffinella was genera. The phylogenetic inference based on SSU the vestibular plate in the vestibulum. However, a rDNA supported the monophyletic status of auto- vestibular plate has also been observed in two other trophic families Baffinellaceae, Falcomonadaceae marine genera Falcomonas and Proteomonas. Follow- (see later), Hemiselmidaceae, , ing the phylogenetic reconstruction based on the and , whereas families single gene tree, cryptophytes possessing vestibular 10 NIELS DAUGBJERG ET AL.

between stages in the heteromorphic life cycle of P. sulcata has not been observed (Hill and Wether- bee 1986). Potvin and Lovejoy (2009) determined the relative amounts of DNA in cells of strains CCMP2045 and CCMP2293. They found that CCMP2045 had twice the amount of DNA compared to CCMP2293. Thus, CCMP2045 likely represents the diplo- and CCMP2293 the haplomorph stages of Baffinella. The two strains had identical SSU rDNA sequences. Unfortunately, the morphology of strain CCMP2293 has not yet been studied in detail but we hypothesize that B. frigidus represents the second marine cryptophyte with a dimorph stage. Future studies will also have to show if a dimorphic stage is characteristic of Falcomonas daucoides. Thus, at pre- sent two out of three cryptophytes with haplo- and diploid stages indicative of sexual reproduction pos- sess vestibular plates. This adds support to the func- tion of the vestibular plate taken part in sexual reproduction. In the freshwater genus Cryptomonas, FIG. 6. Schematic illustrations of Baffinella frigidus gen. et sp. dimorphic species have also been described (Hoef- nov. in ventro-lateral view. (A) Illustration showing different sizes Emden and Melkonian 2003). However, here sexual of the oblique rows of hexagonal periplast plates and length of furrow (fu). The vestibular plate (vp) and the ventral (vf) and reproduction takes place without a vestibular plate. dorsal flagella (df) are also shown. (B) Illustration showing posi- Furrow-gullet complex. The morphology and exten- tion of major cell organelles: nucleus (N), bisected pyrenoid (p), sion of the gullet-furrow complex has been empha- starch sheet (s), nucleomorph (nm), large (le), and small (se) sized as a diagnostic feature in cryptophyte ejectosomes and chloroplast (c). The slightly overlapping peri- taxonomy (e.g., Butcher 1967, Clay et al. 1999). plast plates and serrated outline of the cell exterior is not shown. Kugrens et al. (1986) listed five different types. However, as it can be seen in Table 1 different types of furrow-gullet complexes are present in the same family (Geminigeraceae and Pyrenomonadaceae) making it difficult to use as a diagnostic character at this taxonomic level. Rather the evolution of the furrow-gullet complex appears little constrained and to be identified by character reversals. Baffinella shares a furrow-gullet complex with the marine genus Geraminigera and the freshwater genera Pyreno- monas and Cryptomonas. Like Falcomonas, most other cryptophytes possess only a furrow. Inner periplast components. The shape and appear- ance of this character is also considered important in delineation of cryptophyte genera (Clay et al. 1999). Periplast plates with a hexagonal outline as in Baffinella also occurs in a number of unrelated genera of both marine (Falcomonas, Proteomonas and FIG. 7. Absorbance spectrum of the water-soluble pigments of Baffinella frigidus gen. et sp. nov. showing a peak at 566 nm, ) and freshwater (, Komma revealing the presence of Cr-PE 566. and Hemiselmis) cryptophytes. Based on the tree topology in Figure 8, the evolutionary history of the shape of the inner periplast components was sug- plates are not closely related and therefore this fea- gested to involve independent origins. ture appears to have developed independently at Cell shape and posterior end. The marked comma least three times (or alternatively lost multiple shape of some Baffinella frigidus cells was due to times). Hill and Wetherbee (1986) speculated on an acute posterior. Under the microscope other the function of the vestibular plate in Proteomonas cells were bluntly rounded. The reason for the sulcata, and suggested that it could be a specialized different outline of the posterior end is receptive area similar to a mating structure seen in unknown. In SEM, the tail-like appendage was some gametes of green algae. In this context, it is sometimes seen to bend completely backwards, interesting that Proteomonas contains a haploid and which indicated a high degree of flexibility. A diploid stage each with different morphotypes. This few other cryptophytes have also been described strongly implies sexual reproduction, but transition with an acute posterior end (e.g., F. daucoides, BAFFINELLA FRIGIDUS, AN ARTIC MARINE CRYPTOPHYTE 11

FIG. 8. Phylogenetic tree of cryptophytes (18 genera) revealing the position of Baffinella frigidus gen. et sp. nov. (CCMP2045, marked in bold face) based on nuclear encoded SSU rDNA gene sequences. Roombia truncata (Katablepharidea) formed the outgroup taxon. The tree topology was based on BA and the robustness of clades was evaluated by PP from BA and BS values from ML analyses (500 replica- tions). Support values are indicated at internodes (PP values ≥0.5/BS values ≥50%). Maximum values (PP = 1 and BS = 100%) are indicted by filled circles. Currently, accepted families are superimposed on the tree and indicated by gray boxes. The types of cryptophyte biliproteins (either Cr-PE (phycoerythrin) or Cr-PC (phycocyanin) are indicated for each taxon. Type species are indicated by arrows. Branch lengths are proportional to the number of character changes. 12 NIELS DAUGBJERG ET AL.

there is no close relationship between these taxa indicating multiple origins of a similar cell out- line in cryptophytes. Position of nucleomorph.InBaffinella, the nucleo- morph was positioned anterior to the pyrenoid. A similar position of the pyrenoid is also seen in Fal- comonas, Teleaulax, , and Chroomonas. These taxa represent four families and contrary to Hoef- Emden et al. (2002) we do not find that this charac- ter was completely congruent with the tree topology in our phylogenetic analyses. Rather, the position of the nucleomorph seemed to have had a complex evolutionary history. Phylogeny of Baffinella frigidus. Baffinella frigidus was related to strain CRY1V from NW Spain (Ria de Vigo) identified as Falcomonas sp. using morphology only (Raho et al. 2014). Only a single nucleotide substitution difference separated the two taxa when comparing their SSU rDNA sequences (1,686 base pairs). Falcomonas sp. was only distantly related to F. daucoides identified using electron microscopy and spectroanalysis (Hill 1991a, Clay and Kugrens 1999). The Spanish strain was grown at 18.5°C, whereas it was shown here that B. frigidus cannot survive at temperatures above 10°C. Despite high sequence similarity, the difference in tempera- ture preferences indicates that the two strains repre- sent markedly different ecotypes. Further studies of CRY1V are needed before the taxonomy of this strain can be completed understood. Why a new family and genus?. From the list of characters and their states (Table 1) CCMP2045 did not fit into any of the families in the cryptophyte classification schemes suggested by Clay et al. (1999) and Clay (2015). Due to the presence of Cr- PE 566, Baffinella frigidus belongs to the order Cryp- tomonadales. This order has one family, the Cryp- tomonadaceae but because the disposition of the nucleomorph (anterior to pyrenoid in Baffinella and between the pyrenoid and the nucleus in Cryp- tomonadaceae), morphology of the pyrenoid matrix (bisected in Baffinella and not traversed in Cryp- tomonadaceae), occurrence of vestibular plate (pre- sent in Baffinella and absent in Cryptomonadaceae) it did not allow for an of Baffinella into the Cryptomonadaceae. These differences were con- sidered sufficient for the establishment of a new family. Similarly, no description of an existing cryp- tophyte genus completely matched the features of FIG. 9. Abiotic factors (temperature, salinity and light inten- Baffinella explaining the need for erecting a new sity) affecting the growth rate of Baffinella frigidus gen. et sp. nov. (A) Growth rates as a function of temperatures ranging from 4°C genus. This was also supported by the distinct lin- to 10°C. (B) Growth rates as a function of salinities ranging from eage containing Baffinella in the SSU rDNA tree 5 to 35. (C) Growth rates as a function of light intensities ranging (Fig. 8). 2 1 from 5 to 100 lmol photons m s . Identical letters in each Light dependent chloroplast color. When grown in of the three figures mark non-significant results from one-way ANOVA analyses. Nunc flasks, the color of the chloroplast in Baffinella either appeared green or red depending on the light intensity provided. Hence, at light levels , prolonga, Teleaulax ≥10 lmol photons m 2 s 1, the chloroplast was spp.). The adaptive value (if any) of the poste- green; whereas at low light (5 lmol pho- rior tail-like appendage remains obscure. Again, tons m 2 s 1), it was red. This was also clearly TABLE 1. Characters and characters states used to delineate cryptophyte genera. Their taxonomy into families is also included. Modified from Clay et al. (1999), Clay (2015), Hill (1991b), Hill and Wetherbee (1986, 1988), Hoef-Emden and Archibald (2016) and Laza-Martınez (2012).

Shape of inner Number, Chloro- Furrow-gullet periplast Number, position of Type of morphology plast Vestibular Vestibular Family Genus complex component Rhizostyle nucleomorph biliprotein of pyrenoid number ligule plate Eyespot Habitat Baffinellaceae Baffinella Furrow and Hexagonal Non-keeled 1, anterior to pyrenoid PE 566 1, bisected by 1 No Yes No Marine fam. nov. gen. nov. gullet periplastidial FRIGIDUS BAFFINELLA cytoplasmic tongue Geminigeraceae Teleaulax Furrow only Sheet Keeled 1, anterior to pyrenoid PE 545 1, not traversed 1 No No No Marine Furrow and Sheet Keeled 1, invagination in PE 545 2, not traversed by 1 No No No Marine gullet nucleus Plagioselmis Furrow only Hexagonal Non-keeled 1, between pyrenoid PE 545 1, not traversed 1 No No No Freshwater, and nucleus Marine Guillardia Gullet only Irregular Keeled 1, anterior to pyrenoid PE 545 1, not traversed 1 No No No Marine sheet-like plates Hanusia Furrow only Sheet Keeled 1, between pyrenoid PE 545 1, not traversed 1 No No No Marine and nucleus Proteomonas Furrow only Hexagonal (1n), Non-keeled 1, between pyrenoid PE 545 1, not traversed 1 No Yes No Marine CRYPTOPHYTE MARINE ARTIC AN , sheet (2n) (1n), and nucleous keeled (2n) Urgorria Furrow only Inner sheet (?) Keeled 1, between pyrenoid PE 545 3, not traversed 1 No No Yes Marine (campy- and nucleus lomorph) Falcomonadaceae Falcomonas Furrow only Hexagonal None 1, anterior to pyrenoid PC 569 1, bisected by 1 No Yes No Marine fam. nov. periplastidial cytoplasmic tongue Chroomonadaceae Chroomonas Gullet only, Rectangular or None 1, anterior to pyrenoid PC 630 or 1–2, traversed by 1–2 No No Yes, but Freshwater sometimes hexagonal PC645 thylakoids not all branched Komma Gullet only Rectangular, None Between pyrenoid and PC 645 1, not traversed 1 No No No Freshwater hexagonal nucleus Pyrenomonadaceae Furrow and Rectangular Keeled 1, in pyrenoid matrix PE 545 1, not traversed 1 No No No Freshwater gullet Rhinomonas Gullet only Hexagonal None 1, in pyrenoid matrix PE 545 1, not traversed 1 No No No Marine Gullet only Inner sheet Keeled 1, in pyrenoid matrix PE 545 1, not traversed 1 No No No Freshwater Cryptomonadaceae Cryptomonas Furrow and Fibrous Non-keeled 2, between PE 566 or 0, 2 (4), not 2 Some No No Freshwater gullet and nucleus traversed Hemiselmidaceae Hemiselmis Gullet only Hexagonal None 1, between pyrenoid Red species: 1, round and 1 No No Yes? Freshwater and nucleus PE 555, traversed by PE 577; thylakoids Blue-green species: PC 615, PC630 aUrgorri is provisionally placed in the family Geminigeraceae. 13 14 NIELS DAUGBJERG ET AL. seen in the light microscope and documented in 30; Daugbjerg and Moestrup 1992a,b, Salmansen Figure S3. Thus, the color of B. frigidus was rather and Daugbjerg, unpub. data). For the picoflagellate cryptic. Analysis of water soluble pigments revealed Micromonas sp., the maximum growth rate was a clear peak around 566 nm indicating the presence found to be 0.55 divisions d 1 at 8°C, when light of phycoerythrin Cr-PE 566 when grown at low light. was not a limiting factor (Lovejoy et al. 2007). Cells grown at 20–100 lmol photons m 2 s 1 Thus, comparing the rates of Baffinella to those of showed no peaks corresponding to a biliprotein other Arctic microalgae revealed that it is grows (data not shown). A study on a species of Rhodomo- slightly slower than Pyramimonas spp. and Micromo- nas (now Pyrenomonas) by Chaloub et al. (2015) nas sp. and is in the mid-range compared to the showed similarly that the amount of phycoerythrin pennate Fragilariopsis. A single study has increased when cells were grown at low light levels addressed the growth rate of a freshwater crypto- (<15 lmol photons m 2 s 1) compared to 50 phyte (Rhodomonas sp.) from the Arctic and pro- and 150 lmol photons m 2 s 1. In addition, vided in situ rates in an ice-covered lake to range Chaloub et al. (2015) showed that the phycoery- from 0.07 to 0.18 divisions d 1 (Vehmaa and Salo- thrin content decreased in Rhodomonas cells nen 2009). approaching the stationary phase. Color changes Future studies. Additional studies should address have also been observed in other cryptophytes. the presumable haplomorph of Baffinella frigidus Butcher (1967) and Pringsheim (1968) both noted (CCMP2293) and focus on ultrastructure of the that when cultures were starved the cell color chan- inner periplast component, the vestibular plate, the ged. In this study, cells were probably not starved in position of the nucleomorph and type of phyco- terms of nutrient supply but rather with respect to biliprotein. In addition, a more complete characteri- available light. Light dependent presence of phyco- zation is needed for a proper identification of strain biliproteins should be taken into account when CRY1V from NW Spain. We doubt that it is a species describing the color of chloroplasts in cryptophytes. of Falcomonas and it should be determined if it rep- Taxonomy of Falcomonas. On the basis of phyloge- resents an ecotype of B. frigidus. Temperature is an netic analyses and the comparative study of pheno- environmental factor that clearly distinguishes the typic characters, we propose here to formally unique ecological niches of CCMP2045 and CRY1V, describe the family Falcomonadaceae. This taxon which otherwise seem to form a single phylospecies. was first suggested by Clay and Kugrens (1999), but The growth rate of B. frigidus when provided light it was illegitimate according to IBCN article 39.1 intensities between 5 and 20 lmol pho- (no diagnosis or description provided). tons m 2 s 1 should be investigated to better Description: Falcomonadaceae fam. nov. Daugb- understand when the chloroplast changes color due jerg to disappearance of Cr-PE 566. Despite this first Furrow only; vestibular plate present; nucleo- polyphasic study, our understanding of the species morph anterior to pyrenoid; pyrenoid traversed by diversity of Arctic marine cryptophytes is still in its cytoplasmic tongue; hexagonal inner periplast infancy. Thus, future studies should address the plates; rhizostyle absent. alpha taxonomy of these small sized flagellates from Currently only one genus with one species in the the vast regions of the Arctic. family: Falcomonas daucoides Hill Autecology. On the basis of the results of the aute- We thank Lis Munk Frederiksen, Ayoe Lucheu€ and Øjvind cological experiments, we concluded that Moestrup for technical assistance. We thank Marie Jose Marti- Baffinella frigidus was stenothermal and euryhaline. neau and W.F Vincent for pigment data. ND thanks the Carls- However, the lower temperature and upper salinity berg Foundation (2012-01-0509) and Brødrene Hartmanns ranges where cells can longer to divide were not Fond (A22920) for equipment grants. We also thank review- determined. Yet, it appears that B. frigidus is well ers for their constructive comments on an earlier version of adapted to the environmental settings of Baffin the manuscript. Bay, where the subsurface water temperature rarely ° – Burki, F., Okamoto, N., Pombert, J.-F. & Keeling, P. J. 2012. The gets above 8 C and the salinity lower than 5 10 evolutionary history of and cryptophytes: phy- despite seasonal melting of sea ice. We are unaware logenomic evidence for separate origins. Proc. R. Soc. B Biol. of other autecological studies addressing the poten- Sci. 279:2246–54. tial growth of Arctic marine cryptophytes when Butcher, R. W. 1967. 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